Species from a broad phylogenetic range of insects have been reported to produce thermal-hysteresis proteins (THPs). THPs are believed to play an important role in many plant and animal species' ability to survive exposure to subzero temperatures. By definition, the equilibrium melting and freezing points of water are identical. However, the presence of thermal-hysteresis proteins lowers the non-equilibrium freezing point of water without lowering the melting point (equilibrium freezing point). Thus when THPs are added to a solution they produce a difference between the freezing and melting temperatures of the solution, and this difference has been termed "thermal-hysteresis".
In the absence of THPs a small (about 0.25 mm diameter) ice crystal that is about to melt at the melting point temperature will normally grow noticeably if the temperature is lowered by 0.01.degree. to 0.02.degree. C. However, if THPs are present, the temperature may be lowered as much as 5.degree. to 6.degree. C. below the melting .point (depending upon the specific activity and concentration of the proteins present) before noticeable crystal growth occurs. Consequently, because of THPs ability to lower the freezing point of aqueous solutions, they are commonly referred to as antifreeze proteins. THPs lower the freezing point of aqueous solutions via a non-colligative mechanism that does not depress the vapor pressure or raise the osmotic pressure of water, as is the case with colligative type antifreezes such as glycerol.
Anti-freeze proteins are believed to exert their effect by adsorbing onto the surface of potential seed crystals via hydrogen bonding. When anti-freeze proteins adsorb onto the ice surface they interfere with the addition of water molecules and force growth of the crystal into many highly curved fronts with high surface free energy. Consequently, growth of the crystal requires the temperature to be further lowered to allow crystal growth to proceed.
Thermal-hysteresis proteins were first discovered, and have been best studied, in marine teleost fishes inhabiting seas where subzero temperatures occur, at least seasonally. In these THP-producing Antarctic fish an ice crystal present in the blood serum that melts at -1.1.degree. C., will fail to grow in size until the temperature is lowered to -2.5.degree. C. (thermal hysteresis=1.4.degree. C.). Thus the fish is protected from freezing in its ice-laden -1.86.degree. C. seawater environment.
Over the last 15 years it has been demonstrated that many insects and other terrestrial arthropods (including certain spiders, mites, and centipedes) also produce thermal-hysteresis proteins. Low levels of thermal hysteresis activity are also quite common in overwintering plants and are present in certain fungi and bacteria.
Thermal-hysteresis proteins (THPs) have been isolated from four species of insects: Tenebrio molitor, the milkweed bug, Oncopeltus fasciatus, the spruce budworm, Choristoneura fumiferina, and D. canadensis. The molecular masses of these THPs range from approximately 14 to 20 kDa. The insect THPs characterized to date do not have a carbohydrate component, nor do they have high percentages of alanine like the type-I fish (flounder) THPs.
The amino acid compositions of representative insect THPs are shown in Table 1, and can be generally characterized as having higher percentages of hydrophilic amino acids (i.e. Thr, Ser, Asx, Glx, Lys, Arg) than the fish THPs, with generally 40-50 mol % of the residues being capable of forming hydrogen bonds.
TABLE 1 ______________________________________ Amino acid compositions (mol %) of representative THPs. Amino 1 2 2 acid (H-1) T-4 T-3 3 4 ______________________________________ Asx 14.3 7.3 5.3 9.5 7.1 Thr 17.2 6.6 2.3 6.0 2.7 Ser 10.3 7.4 11.1 13.0 30.5 Glx 5.2 8.9 12.4 11.0 12.3 Pro 2.6 5.9 0.0 5.0 0.0 Gly 6.6 8.3 11.4 15.0 20.0 Ala 8.4 14.3 5.0 8.0 6.8 1/2Cys 15.9 0.0 28.0 6.0 0.0 Val 1.7 11.5 2.3 3.0 3.0 Met 0.2 4.8 0.0 0.0 0.0 Ile 1.5 7.1 1.0 1.2 1.9 Leu 1.9 0.0 2.2 6.5 3.1 Lys 3.4 6.8 15.4 3.1 7.5 Arg 4.8 2.6 0.0 8.0 0.0 Tyr 3.9 2.3 0.0 1.0 2.0 Phe 0.0 3.9 0.0 2.2 1.1 His 1.9 1.9 3.1 0.0 2.3 ______________________________________ 1 = Dendroides canadensis; 2 = Tenebrio molitor; 3 = Choristoneura fumiferana; 4 = Oncopeltus fasciatus
Several of the insect THPs have significant amounts of cysteine. In particular 16 mole % of the amino acid residues of the THPs of D. canadensis are cysteine, approximately half of which are involved in disulfide bridges. Treatment with dithiothreitol, which reduces these disulfide bonds, or alkylation of free sulfhydryls, results in complete loss of thermal-hysteresis activity.
The thermal-hysteresis activity present in the hemolymph (the circulatory fluid of insects) of Dendroides canadensis insects in mid-winter averages 3.degree.-6.degree. C. with some individuals having as much as 8.degree.-9.degree. C. This activity is significantly greater than that which is present in the blood of polar fish; the maximal activity achievable with very high concentrations of the fish THPs is about 1.7.degree. C. FIG. 1 compares the activities of purified THPs from two species of fish and two species of insects: 1=Dendroides canadensis THP, the most active THP currently known (solid squares); 2=Tenebrio molitor THP (solid circles); 3=activity of the most active fish THP, i.e. Antarctic eelpout and THP of Antarctic nototheniids (open squares); 4=activity of the least active fish THP, from cod Gadus morhua (open circles). The maximal activity of the most active fish and T molitor THPs are similar, however, the THPs of D. canadensis have a greater specific activity and a much greater maximal activity than any other known THP. In addition as will be described later, the Dendroides canadensis THPs can be activated by the presence of certain other proteins to produce even greater levels of thermal hysteresis activity.